Figure 4.

Overexpression of nsP3 constructs of different lengths reveals hierarchy of FGDF motif binding in vivo. HEK293 cells were transfected with constructs expressing EGFP fused to the G3BP-binding sequences of SFV nsP3 in the context of (a) only 36 residues, (b) the HVD or (c) the full-length nsP3 protein. In each panel, cells were mock transfected or transfected with pEGFP alone or the construct encoding the WT, F3A_N, F3A_C or F3A_NC sequence. Cell lysates were prepared 24 h after transfection and immunoprecipitated with anti-GFP, separated by SDS–PAGE and transferred to PVDF membranes. Blots of IPs and total lysates were probed for GFP, G3BP-1 or actin. In the lower panels, densitometry analysis was performed on the intensities of G3BP-1 bands in IPs in the upper panels and expressed relative to the appropriate WT construct. Data are averages from at least two experiments and error bars are standard deviations.